Protease-activated receptors (PARs) are distinct G protein-coupled receptors (GPCRs) that allow cells to sense specific proteases in their environment. PARs are emerging as attractive therapeutic targets for several diseases, including cardiovascular diseases, arthritis, colitis, asthma, neurodegenerative conditions and cancer. Individual PARs are able to activate pathways that confer both barrier disruptive, proinflammatory signaling, as well as anti-inflammatory barrier protective signaling pathways. How this selective signaling is mediated is poorly understood, and represents a significant impediment to the development of effective agonists and antagonists targeting PARs for therapeutic uses. A better understanding of the nature of the protease environment that induces differential PAR signaling, and the molecular mechanisms involved in protease activation of PARs are critical. We have discovered that two membrane-anchored serine proteases, Testisin and Matriptase, are cell-specific endogenous activators of PAR2, and likely modulate localized spatial and temporal signaling of PAR2 in endothelial and epithelial cells, respectively. Our data suggests that the GPI anchored serine protease, Testisin, is a proangiogenic factor that can activate PAR2 in the microvasculature, and facilitate capillary growth important for angiogenesis. We also find that the transmembrane serine protease, Matriptase, may sustain a PAR2 dependent signaling pathway important for maintaining the intestinal epithelial barrier. The goal of the research plan is to define the activities of these PAR2-activating membrane serine proteases and to develop a novel prodrug strategy for both detecting and targeting their activities. The research plan will utilize in vitro cultures in concert with in vivo studies in mice to test the following aims: 1) to determine the contribution of Testisin to PAR2 signaling during angiogenesis, 2) to determine the role of PARs in regulating in regulating Prostasin->Matriptase mediated intestinal epithelial barrier closure, and 3) to reengineer anthrax toxins to target PAR2-activating membrane-anchored serine proteases on the cell surface.